Chromatography Systems

Abstract

Disclosed is a chromatography system configured to perform automated chromatography column efficiency tests on a particular column to determine efficiency parameters such as h and As, and optionally further provide guidance to a user regarding column efficiency, the system having a controller configured to automatically access data to enable calculation of column compression plate height, said data being derived from stored parameters of at least the particular column and the resin to be used in said column, and to calculate the efficiency of the column from said data.

Description

[Technical field]

[0001] The present invention relates to a liquid chromatography system configured to operate with at least one column for the purification of a sample containing a target product when the sample is fed into the liquid chromatography system. In particular, the present invention relates to a method for the preparation and qualification of packed columns, and for column efficiency testing. [Background technology]

[0002] An important factor in process chromatography is the binding capacity of a chromatography column for a solute. The binding capacity directly impacts the productivity and cost of a chromatography process. The binding capacity is defined either in terms of the dynamic / breakthrough capacity or as the maximum binding capacity. The dynamic capacity depends on the conditions under which a solution flows through a column packed with a chromatographic medium and can be expressed as the ratio of the column volume to the feed flow rate, the so-called residence time. The maximum binding capacity represents the breakthrough capacity of the column if the residence time were infinitely long.

[0003] When validating a process used in a chromatography system having a single or several chromatography columns, it is important that the results from the process are predictable and repeatable without deviation from the process specifications. In a single column system, when changing columns, it is important that the properties of the new column are of the same type and perform the same, i.e., a column with identical or nearly identical properties is needed to achieve the desired results in the validated process.

[0004] In sequential chromatography, several columns are connected in an arrangement that allows them to operate in series and / or parallel, depending on the requirements of the method. Thus, all columns can in principle be run simultaneously, but with slightly modified method steps. The procedure can be repeated, so that each column is loaded, eluted, and regenerated multiple times in the process. Compared to "conventional" chromatography, where a single chromatography cycle is based on several sequential steps, such as sample loading, washing, elution, stripping, clean-in-place (CIP), and re-equilibration, in multiple-column-based sequential chromatography, all these steps are performed simultaneously, but each on a different column, before the column can be used in another batch.

[0005] To achieve optimal results, columns in a sequential chromatography should be identical or nearly identical. The same is true when replacing columns with a single chromatography column used in a validated process. If the difference in column performance is too great, the process will operate outside the validated performance range.

[0006] Continuous chromatography is an example of a periodic counter current process because periodically all chromatographic columns comprising the system are moved simultaneously in the opposite direction to the sample flow. The apparent movement of the columns is achieved by appropriate redirection of the inlet and outlet flows to and from the columns.

[0007] Historically, the key elements for a reliable continuous process were: 1) the quality of the columns used, and more specifically, the similarity or even identity between the columns; 2) constant supply composition, and 3) Hardware reliability, e.g. pumps delivering a constant flow, valves functioning, etc. It is.

[0008] If the columns are not identical, the theoretical calculations typically used to design continuous chromatographic processes will be invalid, making it difficult to design an efficient and robust continuous chromatographic process. The same arguments apply when feed concentrations and flow rates vary over time in unpredictable ways.

[0009] Therefore, when considering scale-up with identical columns, it is important to have reliable pumps in the system. However, packing the column with chromatography media is a very complicated task to obtain repeatable results. Even small differences in the number of plates or other packing characteristics can have a very large effect on the final results. Furthermore, the capacity of the chromatography resin typically changes during the life / use of the resin, so the process conditions selected for fresh resin / media may not apply to a resin that has been used several times. Also, when the feed solution concentration changes, it becomes even more complicated to design an efficient continuous chromatography process that will always operate at its optimum conditions. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Application Note 28-9372-07AA, GE Healthcare Summary of the Invention [Problem to be solved by the invention]

[0011] It is an object of the present disclosure to provide methods, and devices configured to execute the methods and computer programs, that seek to mitigate, alleviate or eliminate one or more of the above-mentioned shortcomings and disadvantages in the art, singly or in any combination. [Means for solving the problem]

[0012] This object is achieved by a liquid chromatography system configured to operate with at least one column and configured to purify a sample containing a target product using a predefined process. - controlling the operation of a chromatography system to perform a predefined process; - Retrieving accessible column data from data storage, the column data being specific to each column; - adapting at least one process parameter of a predefined process for each column based on the column data; Thereby, a predefined process is adapted for each column to obtain the target product and maintain the performance of the liquid chromatography system; The controller is configured to:

[0013] The advantage is that more stable, repeatable results are obtained from the liquid chromatography process because process parameters can be adapted to compensate for manufacturing variability when each column is manufactured.

[0014] Further objects and advantages can be attained by those skilled in the art from the detailed description. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 illustrates a schematic of a bioprocess purification system designed to purify a target product using liquid chromatography. [Diagram 2] FIG. 1 illustrates continuous chromatography with any number of columns based on simulated moving bed technology. [Figure 3a] FIG. 1 illustrates the principle of three-column chromatography. [Figure 3b] FIG. 1 illustrates the principle of three-column chromatography. [Figure 3c] FIG. 1 illustrates the principle of three-column chromatography. [Figure 4] FIG. 1 illustrates a single-column liquid chromatography system. [Diagram 5] 5 is a simplified flow chart for the liquid chromatography system of FIG. 4 equipped with a pressure sensor. [Figure 6] 5 is a simplified flow chart for the liquid chromatography system of FIG. 4 with only a system pressure sensor. [Figure 7] 5 is a simplified flow chart for the liquid chromatography system of FIG. 4 with a virtual pressure sensor. [Figure 8] FIG. 1 illustrates the concept of assigning columns to a type. [Figure 9] FIG. 1 illustrates data flow within a system adapted to manufacture columns and liquid chromatography systems. [Figure 10] 1 is a flow chart illustrating a method for manufacturing a column for a liquid chromatography system. [Figure 11] 1 is a flow chart illustrating a method for controlling a liquid chromatography system configured to operate with at least one column. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Chromatography systems are designed to purify a target product (protein, biomolecule derived from cell culture / fermentation, natural extract) using at least one packed column of resin to form a purification process. Each column is switched between loading and non-loading steps, such as washing and elution.

[0017] 1 shows a schematic of a bioprocess purification system 10 configured to purify a target product using a separation process. The bioprocess purification system includes multiple steps involving cell culture 11, retention 12, uptake 13, viral inactivation 14, polishing 15, and delivery 16.

[0018] The cell culture step 11 may be a perfusion type culture, which includes continuous addition of nutrients for cell growth in perfusion culture, and continuous removal of products and waste products through drainage and filtration, for example, using an alternating tangential flow filtration (ATF) filter installation. This step may include process control over viable cell density (VCD), and the next step in the process starts when the VCD reaches a predetermined value. The VCD can be controlled by adapting the components of the cell culture medium fed to the culture, or by adding some components directly to the culture. Alternatively, the cell culture is batch type.

[0019] The sample containing the target product is utilized in a cell-free extraction process, for example, by filtration, centrifugation, or another technique.

[0020] The holding step 12 is an optional step depending on the needs of the process, for example if a filter is in-line before the intake step 13. This step includes process control over weight, and the next step in the process begins when a predetermined volume value is reached, or alternatively after a certain time period, or when a predetermined mass is reached. The holding step can be used both to recover a certain amount of filtered feed from a perfusion cell culture or to recover a certain amount of filtered feed from a batch culture.

[0021] The intake step 13 comprises at least one chromatography column that may have a filter in-line before the intake step. The intake step 13 may comprise a continuous chromatography setup as illustrated in FIG. 2, which may be run as a cyclic countercurrent chromatography with a continuous feed of sample from the cell culture step 11, either directly or via a holding step 12, containing the target product. The intake step includes one or more batch elutions, with process control using an in-line UV sensor to account for variations in feed concentration and resin capacity. The next step is initiated when a predetermined quantity value (e.g., volume, mass, or time) is reached.

[0022] In viral inactivation step 14, different options for viral inactivation are available depending on the process needs. One option is to use batch mode at low pH for 30-60 minutes in a hold-up tank. This step may include process controls for volume, time, temperature, and pH. The next step begins when the predetermined time is reached.

[0023] The polishing step 15 can be straight through processing (STP) with connected batch steps or continuous chromatography with continuous loading steps, or a combination of these. The flow rate is adjusted to the perfusion rate required by the production cells, meaning that the flow rate is determined by the preceding step. The steps may include process controls for UV, flow, and volume, and the next step starts when a predefined volume and amount is reached, or alternatively when a timeout is reached.

[0024] The delivery step 16 may be a virus removal step, such as a virus filter, prior to the ultrafiltration step. The delivery step may be used as a concentration step for batch addition of sample from the polishing step. The delivery step may include continuous or batch delivery of product and may include continuous or batch removal of waste. This step may include process control over pH, conductivity, absorption rate, volume, and pressure, and delivery is achieved when a predetermined product concentration is reached within a predefined environment.

[0025] The automation layer 17 is used to handle decision points for the next step in the process. Different types of sensors (not shown), both in-line and off-line sensors, are incorporated into the process flow to monitor different parameters that can be used to provide data to the automation layer 17 that could be used to handle the decision points. Sensors include, but are not limited to, measuring flow only, VCD, weight, pressure, UV, volume, pH, conductivity, absorbance, etc.

[0026] It should be noted that UV absorption is one example of a parameter that can be monitored to detect the composition of the sample being purified, however, other parameters operating in other frequency bands such as IR, fluorescence, X-ray, etc. may also be used.

[0027] The capture step 13 may include a continuous chromatography facility 20, as illustrated in Figure 2, or a single column 240, as illustrated in Figure 4. Continuous chromatography supports process intensification by reducing footprint and improving productivity. In addition, continuous chromatography is particularly suitable for the purification of unstable molecules, as the short process time helps ensure the stability of the target product.

[0028] In Fig. 2, a sample containing a target product is fed into the continuous chromatography 20 via inlet 21 and the eluted target product is available at outlet 22. The continuous chromatography 20 comprises a number of columns A, B, N, each column being provided with a column inlet 23 and a column outlet 24. The column inlet 23 and the column outlet 24 of each column are connected to a valve system 25 configured to cyclically connect the columns to the inlet 21 and the outlet 22 to achieve continuous purification of the target product. An example of a system configuration having three columns is described with respect to Figs. 3a-c.

[0029] The continuous chromatography 20 is further provided with a buffer inlet 26 and a waste outlet 27 to enable it to perform the necessary tasks. An in-line sensor 28 may be provided after the column outlet 24 of each column or may be assigned to the process flow and integrated in the valve system 25. Important parameters such as UV are measured to control the process, as explained below. Another in-line sensor 28' may be provided before the column inlet 23 of the missing column to enable the performance of each column to be directly evaluated. An in-line inlet sensor 26 may also be provided to monitor the composition of the sample fed into the continuous chromatography 20.

[0030] The continuous chromatography may also include off-line sensors 29 that are designed to extract material from the process and subsequently evaluate selected parameters before the material is disposed of as waste.

[0031] Continuous chromatography comprises at least two, e.g. at least three columns, the principle of operation in a three-column (3C) installation is described with reference to Figures 3a-c. The 3C installation is characterized by two parallel flows, i.e. one for the loading of the two columns in the load zone and another for the non-loading steps, e.g. elution and regeneration of the third column.

[0032] In Figure 3a, illustrating step 1, columns A and B are in the load zone. Column A can be overloaded without losing sample, and column B captures the breakthrough from column A. In this way, utilization of the resin binding capacity is maximized.

[0033] In Figure 3b, illustrating step 2, within the load zone, the overloaded column A is switched so that column B becomes the first column and column C becomes the second column. The overloaded column A is then passed through non-loading steps such as elution and regeneration in a parallel workflow.

[0034] In Figure 3c, illustrating step 3, the overloaded column B in the load zone is switched. Then, column C becomes the first column and column A becomes the second column in the load zone, while in a parallel workflow column B is passed through for elution and regeneration. These three steps are repeated cyclically until the required target product volume, mass, or amount is reached (or the resin life is reached and the column needs to be repacked or replaced).

[0035] The sequential chromatography installation illustrated in FIG. 2 may utilize more than three columns, and the same principles apply in a four-column (4C) installation. However, the non-loading step may be the limiting factor in a 3C installation, and may be split into two columns and run in parallel using a third flow path in a 4C installation. In a 4C installation, the loading and non-loading steps can be balanced. More columns increase the flexibility of the system, but the complexity of the valve system 25 becomes increasingly cumbersome. However, some sequential chromatography installations have more than 16 columns.

[0036] 4 shows a schematic of one embodiment of a chromatography system 190 that includes two three-way input valves 160 and 161 that are configured to select input fluid from fluid sources A1, A2, B1, B2 for two system pumps 150 and 151. The chromatography system 190 includes: A pressure sensor 200 for recording the system pressure in the flow path after the system pump; A mixer 210 to ensure proper mixing of the fluids delivered by the pump.

[0037] These correspond to the cell culture block 11 illustrated in FIG.

[0038] The system is an injection valve 220 for injecting the sample into the fluid path; a column connection valve 230 for selectively connecting / disconnecting a column 240 in the fluid path; a pre-column pressure sensor 235 and a post-column pressure sensor 236; an ultraviolet (UV) monitor 250 for detecting the output from the column; A conductivity monitor 260; and pH Monitor 265 and It further comprises:

[0039] These correspond to the capture blocks 13 illustrated in FIG.

[0040] The system is an output selection valve 270 with two or more output positions, as indicated by dashed line 160 and corresponding to the delivery block 16 in FIG. 1, connected to, for example, a fraction collector 280, a waste receptacle, or the like; a system controller 300, connected to pumps and valves for controlling liquid flow through the system, as shown by dashed line 170, corresponding to automation block 17 of FIG. 1, and to sensors and monitors for monitoring the flow, connections illustrated by dotted line 310; It further comprises:

[0041] The chromatography system of Figure 4 represents a general example of how a single column chromatography system may be designed, other embodiments may be of different designs including two or more of some of the components, and potentially lacking some of these components, such as components corresponding to retention 12, viral inactivation 14, and polishing 15 as illustrated in Figure 1.

[0042] FIG. 5 is a simplified flow chart for the liquid chromatography system 190 according to FIG. 4. In FIG. 5, the flow paths have been straightened out and some components have been removed for a simpler view. In FIG. 5, the system controller is shown connected only to the pump 150, the pressure sensor 200, the pre-column pressure sensor 235, and the post-column pressure sensor 236, but may be connected to other components as described above. In FIG. 5, the system includes both the pre-column pressure sensor 235 and the post-column pressure sensor 236, whereby the column pressure is measured directly by the pre-column sensor 235 and the column differential pressure is measured by subtracting the pressure recorded by the post-column sensor 236 from the column pressure.

[0043] As briefly mentioned above, some systems do not have a pressure sensor separate from the system pressure sensor 200. FIG. 6 is a simplified flow chart of such a liquid chromatography system 190 with one single pressure sensor 200 for recording the system pressure. As mentioned above, pressure control in such a system relies solely on the system pressure recorded by the sensor 200. FIG. 7 is a simplified flow chart of a liquid chromatography system according to an embodiment of the present invention, in which a controller 300 is arranged to estimate a pre-column pressure based on the recorded system pressure, characteristics of the flow path, and the viscosity and flow rate of the fluid in the system. The estimated pre-column pressure may be referred to as a "virtual pressure sensor" as shown diagrammatically in FIG. 7 by a light dashed line.

[0044] According to one embodiment, the calculation of the virtual pressure signal may be based on Bernoulli's equation for the pressure drop in the flow path. Flow path ΔΡ[MPa]=0.000000000679*L*Q*V / D <4> ,however L = length [mm] D=diameter [mm] Q=Flow rate [ml / min] V=viscosity [cP]

[0045] By providing the length and diameter of the flow path, as well as the viscosity of the liquid in the system to the system controller, it may be arranged to calculate the pressure drop caused by the flow path to the column at the current flow rate. In some systems, the length and size of the flow path between the system pressure sensor 200 and the column 240 may be standardized, so that predefined parameters can be used in the calculation. In other systems (which is the most common situation), the flow paths between components in the chromatography system are user-defined paths, so that a user of the system must enter the parameters to be used in a user interface.

[0046] According to one embodiment, the majority of the flow path between the system pressure sensor 200 and the column 240 may be assumed to consist of capillaries of the same diameter, and the flow path properties may then be estimated as the total length of the tubing, thereby excluding contributions from other components such as valves or the like from the calculation. In other embodiments, contributions from valves or the like in the flow path are taken into account and may be system defined, while the tubing or the like is user defined. Note that if the flow path comprises sections of different sizes (e.g., tubing with different internal diameters), the pressure drop over each section must be calculated separately and finally added together to provide the total pressure drop.

[0047] When the pressure drop in the flow path up to column 240 has been estimated by the calculation above, the virtual pre-column pressure is calculated by subtracting the pressure drop from the system pressure recorded by system pressure sensor 200.

[0048] (Example) If the system pressure is 5 bar and the calculated pressure drop over the flow path is 2 bar, the calculated virtual pre-column pressure is estimated to be 3 bar.

[0049] All pressure contributions after the virtual pressure sensor are automatically corrected, since they directly affect the measured system pressure. Thus, for example, if a flow restrictor is added or removed, the measured system pressure will change, and so will the calculated pre-column pressure. Changes in the flow path between the system pressure sensor and the column must be taken into account in the estimation.

[0050] According to one embodiment, if the viscosity is not known, the controller may assume that water is being used and therefore the viscosity can be estimated for different temperatures using the following equation, where viscosity is known: V[cP]=A×10B / (TC), where T = temperature [K], A = 0.02414, B = 247.8K, and C = 140K.

[0051] In a real situation, there may be several factors that may affect the accuracy for the virtual pressure estimation. If the viscosity of the liquid is not known and it is assumed to be water, but has a higher viscosity, the estimated value ΔP for the flow path will be too low. The calculated value for the virtual pressure signal will then be higher than the actual value, which will cause a pressure alarm before the actual pressure for the column becomes too high. This is even if other components in the flow path (mixers, valves, etc.) generate some back pressure. As a result, for liquids with a viscosity lower than water, this estimation will result in a virtual pre-column pressure that is lower than the actual pressure. However, such liquids are often used in high pressure columns, and such columns often withstand higher pressures than they are usually used with, so high accuracy of the pressure signal is not necessary. According to one embodiment, the system is arranged to estimate the column differential pressure by using the same principle for the flow path after the column, and the virtual post-column pressure can be estimated and used to calculate the virtual column differential pressure.

[0052] As mentioned, the virtual pre-column pressure and column differential pressure can be used to control the operation of the chromatography system, for example, by monitoring the pressure with respect to predefined or user-defined pressure limits, or by running the chromatography system at a predefined column pressure or a similar pressure.

[0053] The present disclosure illustrates a chromatography process configured to operate with at least one column and configured to purify a sample containing a target product using a predefined process, which may be a generic process, a validated process, or a specialized process, and may be predefined from the manufacturer or generated by an end user.

[0054] 9 illustrates data flow within a system adapted to manufacture columns and liquid chromatography systems. As explained in more detail below, column data is generated during column manufacture and stored in a database dB accessible to the chromatography system 90. Data flow is shown as dotted lines and control signals are shown as dashed lines.

[0055] The liquid chromatography system comprises a controller 91 configured to control operation of the chromatography system to run a predefined process, to retrieve accessible column data from the data storage dB, the column data being specific to each column, and to adapt at least one process parameter of the predefined process for each column based on the column data, whereby the predefined process is adapted for each column to obtain a target product and maintain performance of the liquid chromatography system.

[0056] The data storage can be a database (integrated within the chromatography system or accessible from a source external to the chromatography system, such as a cloud-based implementation). Another alternative is to store the column data on the individual columns, for example in the form of a memory chip and communication with the chromatography system via RFID.

[0057] The process parameters include the pressure applied to each column, the flow rate of the sample into the column, the flow rate of the retentate out of the column, and / or the processed volume of the sample per time period (column volume / time).

[0058] According to some embodiments, the liquid chromatography system is configured to operate with a single column for purification of a sample, and according to some embodiments, the liquid chromatography system is configured to operate with at least three columns for sequential purification of a sample.

[0059] 8 illustrates the concept of assigning columns to types 400 (also referred to as families). Each individual column 401-1, 401-2, 401-n belongs to a particular type 400 that has predefined production parameters associated with it, in this example, that define ranges of different physical properties of used column components and different process characteristics used to manufacture the column.

[0060] The column comprises a container for holding the resin and a filter, and the column components may include hardware specific properties, physical dimensions of the container (such as the height of the container), material properties of the resin such as particle size and distribution, physical properties of the filter, etc.

[0061] Process characteristics such as resin bed height, pressure boundaries, flow specifications, etc. are relevant to the manufacturing process of the column. The resin bed height can be the actual height or the vessel height.

[0062] The production parameters may further include the actual volume of resin in each column.

[0063] In some exemplary embodiments, the liquid chromatography system further comprises sensors 92a, 92b adapted to read sensor parameters, and the adaptation of the at least one process parameter is further based on the sensor readings. In some embodiments, the sensor readings include any of UV, flow rate, and pressure.

[0064] In some embodiments, the production parameters further include the actual volume of resin in each column.

[0065] A database, accessible by the system, may contain historical data for each individual column and / or for columns of the same particular type (i.e., same family) that have already been used in the liquid chromatography system for purification of samples. Historical data information from the controller is stored in the database for this purpose.

[0066] 10 is a flow chart illustrating a method for manufacturing a column for a liquid chromatography system. The present disclosure also includes a method for manufacturing a column for a liquid chromatography system, the column having an inlet and an outlet, and also having a container for holding a resin. The method includes the steps of: 1) Selecting the type of column that is configured to be used in the process for purification of a sample containing a target product, step S2. 2) Select a resin (which is a media) based on the type of column, the resin having media properties, step S3. Media properties include measured and calculated properties of the media intended to be used in the column. Media parameters are affected by the media manufacturing process and the tolerances of different media components used in manufacturing the media. 3) Select a container (i.e., hardware) to hold the resin, the container having hardware characteristics, step S4. Hardware characteristics include measured and calculated characteristics of different components used to manufacture the column that affect the function of the column, such as physical dimensions (with tolerances) and filter characteristics (if included). 4) Packing the resin to form a resin bed in the vessel using pressure based on the type of column to determine the height of the resin bed, step S5. 5) determining production parameters based on the media characteristics and hardware characteristics to define column data for the column, step S6, and storing the column data in a data storage accessible to the liquid chromatography system. The data storage may be integrated in the column or a database accessible to the chromatography system, as described above.

[0067] In some examples, the column further comprises hardware in the form of a top filter disposed between the inlet and the resin bed, and the method further comprises selecting a top filter having top filter characteristics based on the column type, and determining production parameters further based on the top filter characteristics to define column data.

[0068] In some examples, the column further comprises hardware in the form of a bottom filter disposed between the outlet and the resin bed, and the method further comprises the steps of selecting a bottom filter having bottom filter characteristics based on the column type, and determining production parameters further based on the bottom filter characteristics to define column data.

[0069] The present disclosure includes a column with an inlet and an outlet, and a container for holding a resin, for a liquid chromatography system, the column being manufactured according to the method described above. A system for manufacturing a column is illustrated in Figure 9, and the output from the system is the column, and column data stored in a database dB accessible to the chromatography system.

[0070] In some examples, the column further comprises hardware in the form of data storage, e.g., a data chip, configured to store the column data, and a communication device configured to communicatively communicate the column data to a liquid chromatography system.

[0071] In some embodiments, the communication device is configured to communicate with the liquid chromatography system using RFID.

[0072] 11 is a flow chart illustrating a method for controlling a liquid chromatography system configured to operate with at least one column. The present disclosure also includes a method for controlling a liquid chromatography system configured to operate with at least one column and configured to purify a sample containing a target product using a predefined process, the method comprising: A) controlling the operation of a chromatography system to run a predefined process, step S12; B) retrieving accessible column data specific to each column from the data storage, step S13; and adapting at least one process parameter of a predefined process for each column based on the column data, step S15; Includes.

[0073] Thereby, a predefined process is adapted for each column to obtain the target product and maintain the performance of the liquid chromatography system from the sample.

[0074] In some examples, each column is of a particular type and the data storage includes historical data for each column and / or multiple columns of the same particular type already used in the liquid chromatography system for purification of the sample, and the method further includes step S13a of adapting at least one process parameter of the predefined process based on the historical data.

[0075] In some examples, the data storage includes column data related to production parameters when producing each column, the production parameters including resin bed height, pressure boundaries, flow specifications, material properties, hardware specific properties, filter properties, and physical dimensions of the vessel, and the method further includes step S13b of adapting at least one process parameter of the predefined process based on the production parameters.

[0076] In some examples, the liquid chromatography system further comprises a sensor adapted to read a sensor parameter, and the method further comprises step S14a of further adapting at least one process parameter based on the sensor reading. The sensor reading may include any of UV, flow rate, and pressure.

[0077] The data storage may be selected to be a database, which may be integrated into the liquid chromatography system. An alternative is to integrate the data storage within each column.

[0078] In addition to the above, preparation and qualification of packed columns is a key step to ensure robustness and safety of both the purification process and the final product. Column efficiency testing plays a central role in verifying and monitoring the performance of packed beds. Even though it cannot be used as a single parameter to predict purity and recovery, it is a quick way to test column and equipment performance before starting the purification process. This test can also be used between runs to check for changes in bed integrity.

[0079] This note provides an overview of the background theory and experimental practices used in column efficiency testing. Test conditions are recommended and key parameters influencing the measured efficiency are discussed to facilitate the development of a robust test protocol.

[0080] Column efficiency testing is the analysis of the residence time distribution of a tracer substance passing through a column. Typical test signals applied to the column are pulse or step signals. To characterize chromatographic columns without interference, the conditions of the tracer substance and the eluent are selected to avoid chemical interactions with the media and disturbances of the fluid flow. The most common type of test signal applied is the pulse function. A small amount of the tracer substance is added to the liquid stream near the column inlet and the spread of this pulse is analyzed when measured as a chromatographic peak at the column outlet.

[0081] Column efficiency is typically defined in terms of two parameters: Peak broadening on a column is described in terms of equivalent theoretical plates (equilibrium stages). The symmetry of a peak is described by the peak asymmetry factor As.

[0082] Peak broadening is typically described in terms of plate number, N, or height equivalent to a theoretical plate (HETP). This concept corresponds to the tanks-in-series model, which reflects the number of equilibrium stages represented by the column.

[0083] A widely used method for evaluating pulse tests (to determine plate number) involves measuring the peak width at half the maximum peak height. This approach is an alternative form of numerical curve integration when applying the method of moments, where the first moment is the mean value and the second moment is the retention volume / time variance. As outlined in Figure 1, the pulse response is plotted against time or elution volume and the half-width is measured and related to the elution time or, preferably, the elution volume at the maximum peak height. The retention time or retention volume measured at the maximum peak height corresponds to the average residence time or volume seen for a symmetric (Gaussian) peak shape. A dimensionless and therefore easy to use parameter for efficiency characterization is the reduced plate height, h. This parameter facilitates comparison of column efficiencies regardless of column length and media particle size.

[0084] Optimal column efficiency typically corresponds to an experimentally determined reduced plate height of h≦3 for the porous media employed in bioprocess chromatography. This efficiency is achieved when testing a fully packed bed with an optimized setup of the column and system under optimal test conditions.

[0085] A detailed description of the column efficiency test is provided in Application Note 28-9372-07AA issued by GE Healthcare, which is hereby incorporated by reference in its entirety.

[0086] In one embodiment of the invention, a chromatography system is configured to perform an automated column efficiency test to determine efficiency parameters such as h and As, and further to provide guidance to the user regarding column efficiency. The automated tests presented herein provide all possible data for calculating the reduced plate height since the software automatically calculates the efficiency of the column using specific parameters for a given column and resin. This provides the possibility to directly know whether a given column is good enough for the intended application. If not, the user is notified so that a more suitable column can be used. Suitable cleaning procedures may be applied, the column may be refilled, the column may be replaced with a new column, or any other necessary measures may be taken. This results in a safer and more robust procedure for the liquid chromatography process since the efficiency of the column can be ensured.

[0087] According to one embodiment, a chromatography system is configured to automatically determine one or more efficiency parameters such as the reduced plate height h and As and based on the results, to guide the user of the system to relevant actions, or alternatively, the system may be configured to automatically initiate some or all relevant actions based on the efficiency parameters.

[0088] In one example, the system may be configured to automatically or semi-automatically initiate one or more of the following actions to improve column efficiency. · When 3 < h < 3.5 (Level 1), a standard column cleaning procedure (e.g., 0.5M NaOH) · When 3.5 < h < 5 (Level 2), an intensive column cleaning procedure (e.g., 1M NaOH, acid, and / or isopropanol) · When 5 < h < 7 (Level 3), the column refilling procedure · h > 7 (Level 4), cancel the process and instruct the user to replace the resin

[0089] After the action is completed, the system is preferably configured to automatically determine one or more efficiency parameters and verify the improved efficiency.

[0090] In an alternative embodiment, the system may be configured to repeat one or more of the above actions two or more times if the desired result is not achieved. The system may be further configured to move to a higher level after a predetermined trial at a certain level.

[0091] Note that the above ranges for different levels may vary depending on the type of resin and the type of column. These ranges may also vary depending on the chromatographic process step in which the column is used. For example, a column prepared for a capture step may have a higher h compared to a column prepared for an intermediate or polishing step.

[0092] The chromatography system may be arranged to retrieve particle size data for the determination of efficiency parameters from a predetermined standard data file in a database or from measurement data of the particle size of a specific resin lot used. Lot-specific particle size data may be determined by separate or interconnected measurement units or, alternatively, retrieved from a database containing lot-specific particle size data, such as a cloud-based database.

[0093] According to one embodiment, the automated column verification further includes determining a column differential pressure to verify the column packing performance. If the determined differential pressure is determined to be too low, there is a high risk of gaps, whereas if the differential pressure is determined to be too high, it may be outside the range of the system pressure. The relevant range for the differential pressure may be retrieved from a column database and / or a system setting.

[0094] The present disclosure further includes a computer program for controlling a liquid chromatography system comprising instructions which, when executed on at least one processor, cause the at least one processor to perform the method according to the method set out above.

[0095] The present disclosure further includes a computer readable storage medium containing a computer program for controlling a liquid chromatography system as defined above. [Explanation of symbols]

[0096] 10. Bioprocess Purification Systems 11 Cell culture 11 Cell Culture Blocks 12 retention 13 Import 13 Import Block 14 Virus inactivation 15 Polishing 16 Delivery 17 Automation layer 20 Continuous Chromatography Equipment 21 Entrance 22 Exit 23 Column inlet 24 Column outlet 25 Valve System 26 Buffer inlet 26 In-line inlet sensor 27 Waste outlet 28 Inline Sensor 28' Inline Sensor 29 Offline Sensors 90 Chromatography Systems 91 Controller 92a, 92b Sensor 110 Dashed Line 130 dashed line 150, 151 System pump 160, 161 Three-way input valve 160 dashed line 170 dashed line 190 Chromatography System 200 Pressure Sensor 210 Mixer 220 Injection valve 230 Column connection valve 235 Pre-column pressure sensor 236 Post-column pressure sensor 240 Single Column 250 Ultraviolet (UV) Monitor 260 Conductivity Monitor 265 pH Monitor 270 Output selection valve 280 Fraction collector 300 System Controller 310 Dotted Line 400 Type 401-1, 401-2, 401-n columns

Claims

1. A chromatography system configured to perform an automated chromatography column efficiency test on a particular column to determine an efficiency parameter and further provide guidance to a user regarding column efficiency, wherein the efficiency parameter is a column compression stage height h, the system having a controller configured to automatically access data to calculate the column compression stage height, the data including at least stored parameters of the particular column and a resin to be used in the column, thereby calculating the column efficiency.

2. 10. The chromatography system of claim 1, wherein the system determines whether the particular column is suitable for use or not suitable for use based on the column compression stage height.

3. 3. The chromatography system of claim 2, wherein if the column is determined to be unsuitable, a suitable column repacking procedure is recommended.

4. 4. A chromatography system as described in any one of claims 1 to 3, further comprising a user interface, wherein the chromatography system is configured to automatically determine the efficiency parameters and present relevant actions on the user interface based on the results, or alternatively, the system may be configured to automatically initiate some or all relevant actions based on the efficiency parameters.

5. The system performs the following actions to improve the column efficiency: When 3<h<3.5 (Level 1), standard column wash procedure 3. When 5<h<5 (level 2), a strong column wash procedure When 5<h<7 (level 3), the column repacking procedure When h>7 (level 4), cancel the process and prompt the user to replace the resin.

13. The chromatography system of claim 1, configured to automatically or semi-automatically initiate one or more of:

6. 6. The chromatography system of claim 5, wherein after one or more of the actions are completed, the system is configured to further automatically determine one or more efficiency parameters and verify any improved efficiency.

7. 7. The chromatography system of claim 5 or 6, wherein the system is configured to repeat one or more of the above actions two or more times if the efficiency parameter is not reached, or at a higher level after a given trial at a lower level.

8. 8. The chromatography system of claim 1, wherein the automated column verification further comprises determining a differential pressure of the column to verify column packing performance.

9. 9. The chromatography system of claim 8, wherein if the determined differential pressure is determined to be too low, the user is informed that there is an increased risk of gaps, but if the differential pressure is determined to be too high, the user is informed that the differential pressure may be outside the pressure range of the system.

10. The chromatography system of claim 9 , wherein the ranges associated with differential pressure are retrieved from a column database and / or a system setting.

11. A method for use in a chromatography system configured to perform an automated chromatography column efficiency test on a particular column to determine an efficiency parameter, and further provide guidance to a user regarding column efficiency, the efficiency parameter being a column compression stage height, h, the method comprising: a) providing a system controller; b) configuring said controller to automatically access data to enable said controller to calculate said column compression stage height, said data including at least stored parameters of said particular column and resin to be used in said column, thereby calculating said column efficiency.

12. The method further comprises any one or more of the following steps: c) configuring said controller to determine whether said particular column is suitable for use or unsuitable for use based on said column compression stage height; d) performing a repacking procedure if the column is determined to be unsuitable; e) configuring said system to further provide a user interface, automatically determine said efficiency parameters, and present recommended associated actions in said user interface based on said determination; or f) taking the following actions to improve the column efficiency: When 3<h<3.5 (level 1), normal column washing procedure 3. When 5<h<5 (level 2), a strong column wash procedure When 5<h<7 (level 3), the column repacking procedure When h>7 (level 4), cancel the process and instruct to replace the column resin. configuring said system to automatically or semi-automatically initiate one or more of The method of claim 11 further comprising:

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